Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and proteinprotein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases. R ecent years have witnessed significant interest in biological applications of novel inorganic nanomaterials such as nanocrystals (1, 2), nanowires (3), and nanotubes (4, 5) with the motivation to create new types of analytical tools for life science and biotechnology. Single-walled carbon nanotubes (SWNTs) are interesting molecular wires (diameter Ϸ1-2 nm) with unique electronic properties that have been spotlighted for future solid-state nanoelectronics (6, 7). Bridging nanotubes with biological systems, however, is a relatively unexplored area, with the exception of a few reports on nanotube probe tips for biological imaging (4), nonspecific binding (NSB) of proteins (8-10), functionalization chemistry for bioimmobilization on nanotube sidewalls (5), and one study on biocompatibility (11).Previously, we and others have shown that the electrical conductance of a nanotube is highly sensitive to its environment and varies significantly with changes in electrostatic charges and surface adsorption of various molecules (12)(13)(14). This research has hinted at possible SWNT-based miniature sensors for detecting biological molecules in fluids. Here, we systematically explore how nanotubes interact with and respond to various proteins in solution, how chemical functionalization can be used to tailor these interactions, and how the resulting understanding enables highly selective nanotube sensors for the electronic detection of proteins. Using atomic force microscopy (AFM) and quartz crystal microbalance (QCM) and electronic transport measurements, we first reveal that proteins in general exhibit a high degree of NSB on nanotubes, a phenomenon undesirable for potential biosensors. We then demonstrate a functionalization scheme involving irreversible adsorption of Tween 20 or triblock copolymer chains on nanotubes to prevent this general NSB, while at the same time enabling the binding of specific proteins of interest that can be detected electronically without the need for labeling. Further, we demonstrate specific detection of mAbs to the human autoantigen U1A, a prototype target of the autoimmune response in patients with systemic lupu...
We have developed an acrylic microfluidic device that sequentially couples liquid-phase isoelectric focusing (IEF) and free solution capillary electrophoresis (CE). Rapid separation (<1 min) and preconcentration (73x) of species were achieved in the initial IEF dimension. Using full-field fluorescence imaging, we observed nondispersive mobilization velocities on the order of 20 microm/s during characterization of the IEF step. This transport behavior allowed controlled electrokinetic mobilization of focused sample bands to a channel junction, where voltage switching was used to repeatedly inject effluent from the IEF dimension into an ampholyte-based CE separation. This second dimension was capable of analyzing all fluid volumes of interest from the IEF dimension, as IEF was 'parked' during each CE analysis and refocused prior to additional CE analyses. Investigation of each dimension of the integrated system showed time-dependent species displacement and band-broadening behavior consistent with IEF and CE, respectively. The peak capacity of the 2D system was approximately 1300. A comprehensive 2D analysis of a fluid volume spanning 15% of the total IEF channel length was completed in less than 5 min.
Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Δψ) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
We demonstrate a label-free peptide-coated carbon nanotube based immunosensor for the direct assay of human serum. A rheumatoid arthritis (RA) specific (cyclic citrulline-containing) peptide, was immobilized to functionalized single-walled carbon nanotubes deposited on a quartz crystal microbalance (QCM) sensing crystal. Serum from RA patients was used to probe these nanotubebased sensors, and antibody binding was detected by QCM sensing. Specific antibody binding was also determined by comparing the assay of two serum control groups (normal and diseased sera), and the native unmodified peptide. The sensitivity of the nanotube-based sensor (detection in the femtomol range) was higher than that of the established ELISA and recently described microarray assay systems, detecting 34.4% and 37.5% more RA patients with anti-citrullinated peptide antibodies than those found by ELISA and microarray respectively. As well as 18.4% and 19.6% greater chance of a negative test being a true indicator of a person not having RA than by either ELISA or microarray respectively. The performance of our label-free biosensor constitutes its application in the direct assay of sera in research and diagnostics.
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